Over the past 2 decades, the prevalence of macrolide-resistant Streptococcus pneumoniae (MRSP) has increased considerably, due to widespread macrolide use. Although macrolide usage has been proposed to be associated with treatment failure in patients with pneumococcal diseases, macrolides may be clinically effective for treating these diseases, regardless of the susceptibility of the causative pneumococci to macrolides. As we previously demonstrated that macrolides downregulate the transcription of various genes in MRSP, including the gene encoding the pore-forming toxin pneumolysin, we hypothesized that macrolides affect the proinflammatory activity of MRSP. Using HEK-Blue cell lines, we found that the supernatants from macrolide-treated MRSP cultures induced decreased NF-κB activation in cells expressing Toll-like receptor 2 and nucleotide-binding oligomerization domain 2 compared to the supernatants from untreated MRSP cells, suggesting that macrolides inhibit the release of these ligands from MRSP. Real-time PCR analysis revealed that macrolides significantly downregulated the transcription of various genes encoding peptidoglycan synthesis-, lipoteichoic acid synthesis-, and lipoprotein synthesis-related molecules in MRSP cells. The silkworm larva plasma assay demonstrated that the peptidoglycan concentrations in the supernatants from macrolide-treated MRSP cultures were significantly lower than those from untreated MRSP cultures. Triton X-114 phase separation revealed that lipoprotein expression decreased in macrolide-treated MRSP cells compared to the lipoprotein expression in untreated MRSP cells. Consequently, macrolides may decrease the expression of bacterial ligands of innate immune receptors, resulting in the decreased proinflammatory activity of MRSP. IMPORTANCE To date, the clinical efficacy of macrolides in pneumococcal disease is assumed to be linked to their ability to inhibit the release of pneumolysin. However, our previous study demonstrated that oral administration of macrolides to mice intratracheally infected with macrolide-resistant Streptococcus pneumoniae resulted in decreased levels of pneumolysin and proinflammatory cytokines in bronchoalveolar lavage fluid samples compared to the levels in samples from untreated infected control mice, without affecting the bacterial load in the fluid. This finding suggests that additional mechanisms by which macrolides negatively regulate proinflammatory cytokine production may be involved in their efficacy in vivo. Furthermore, in this study, we demonstrated that macrolides downregulated the transcription of various proinflammatory-component-related genes in S. pneumoniae, which provides an additional explanation for the clinical benefits of macrolides.

Bacterial persisters represent a small subpopulation that tolerates high antibiotic concentrations without acquiring heritable resistance, and it may be generated by environmental factors. Here, we report the first antibiotic persistence mechanism in Streptococcus pneumoniae, which is induced by oxidative stress conditions and allows the pneumococcus to survive in the presence of fluoroquinolones. We demonstrated that fluoroquinolone persistence is prompted by both the impact of growth arrest and the oxidative stress response induced by H2O2 in bacterial cells. This process protected pneumococci against the deleterious effects of high ROS levels induced by fluoroquinolones. Importantly, S. pneumoniae develops persistence during infection, and is dependent on the oxidative stress status of the host cells, indicating that its transient intracellular life contributes to this mechanism. Furthermore, our findings suggest persistence may influence the outcome of antibiotic therapy and be part of a multistep mechanism in the evolution of fluoroquinolone resistance. IMPORTANCE In S. pneumoniae, different mechanisms that counteract antibiotic effects have been described, such as vancomycin tolerance, heteroresistance to penicillin and fluoroquinolone resistance, which critically affect the therapeutic efficacy. Antibiotic persistence is a type of antibiotic tolerance that allows a bacterial subpopulation to survive lethal antimicrobial concentrations. In this work, we used a host-cell infection model to reveal fluoroquinolone persistence in S. pneumoniae. This mechanism is induced by oxidative stress that the pneumococcus must overcome to survive in host cells. Many fluoroquinolones, such as levofloxacin and moxifloxacin, have a broad spectrum of activity against bacterial pathogens of community-acquired pneumonia, and they are used to treat pneumococcal diseases. However, the emergence of fluoroquinolone-resistant strains complicates antibiotic treatment of invasive infections. Consequently, antibiotic persistence in S. pneumoniae is clinically relevant due to prolonged exposure to fluoroquinolones likely favors the acquisition of mutations that generate antibiotic resistance in persisters. In addition, this work contributes to the knowledge of antibiotic persistence mechanisms in bacteria.

Natural transformation plays an important role in the formation of drug-resistant bacteria. Exploring the regulatory mechanism of natural transformation can aid the discovery of new antibacterial targets and reduce the emergence of drug-resistant bacteria. Competence is a prerequisite of natural transformation in Streptococcus pneumoniae, in which comCDE operon is the core regulator of competence. To date, only ComE has been shown to directly regulate comCDE transcription. In this study, a transcriptional regulator, the catabolite control protein A (CcpA), was identified that directly regulated comCDE transcription. We confirmed that CcpA binds to the cis-acting catabolite response elements (cre) in the comCDE promoter region to regulate comCDE transcription and transformation. Moreover, CcpA can coregulate comCDE transcription with phosphorylated and dephosphorylated ComE. Regulation of comCDE transcription and transformation by CcpA was also affected by carbon source signals. Together, these insights demonstrate the versatility of CcpA and provide a theoretical basis for reducing the emergence of drug-resistant bacteria. IMPORTANCE Streptococcus pneumoniae is a major cause of bacterial infections in humans, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Like most streptococci, S. pneumoniae is naturally competent and employs this ability to augment its adaptive evolution. The current study illustrates CcpA, a carbon catabolite regulator, can participate in the competence process by regulating comCDE transcription, and this process is regulated by different carbon source signals. These hidden abilities are likely critical for adaptation and colonization in the environment.

Drug-resistant bacteria are a serious threat to human health as antibiotics are gradually losing their clinical efficacy. Comprehending the mechanism of action of antimicrobials and their resistance mechanisms plays a key role in developing new agents to fight antimicrobial resistance. The lipopeptide daptomycin is an antibiotic that selectively disrupts Gram-positive bacterial membranes, thereby showing slower resistance development than many classical drugs. Consequently, it is often used as a last resort antibiotic to preserve its use as one of the least potent antibiotics at our disposal. The mode of action of daptomycin has been debated but was recently found to involve the formation of a tripartite complex between undecaprenyl precursors of cell wall biosynthesis and the anionic phospholipid phosphatidylglycerol. BceAB-type ABC transporters are known to confer resistance to antimicrobial peptides that sequester some precursors of the peptidoglycan, such as the undecaprenyl pyrophosphate or lipid II. The expression of these transporters is upregulated by dedicated two-component regulatory systems in the presence of antimicrobial peptides that are recognized by the system. Here, we investigated whether daptomycin evades resistance mediated by the BceAB transporter from the bacterial pathogen Streptococcus pneumoniae. Although daptomycin can bind to the transporter, our data showed that the BceAB transporter does not mediate resistance to the drug and its expression is not induced in its presence. These findings show that the pioneering membrane-active daptomycin has the potential to escape the resistance mechanism mediated by BceAB-type transporters and confirm that the development of this class of compounds has promising clinical applications.IMPORTANCEAntibiotic resistance is rising in all parts of the world. New resistance mechanisms are emerging and dangerously spreading, threatening our ability to treat common infectious diseases. Daptomycin is an antimicrobial peptide that is one of the last antibiotics approved for clinical use. Understanding the resistance mechanisms toward last-resort antibiotics such as daptomycin is critical for the success of future antimicrobial therapies. BceAB-type ABC transporters confer resistance to antimicrobial peptides that target precursors of cell-wall synthesis. In this study, we showed that the BceAB transporter from the human pathogen Streptococcus pneumoniae does not confer resistance to daptomycin, suggesting that this drug and other calcium-dependent lipopeptide antibiotics have the potential to evade the action of this type of ABC transporters in other bacterial pathogens.

The short generation time of many bacterial pathogens allows the accumulation of de novo mutations during routine culture procedures used for the preparation and propagation of bacterial stocks. Taking the major human pathogen Streptococcus pneumoniae as an example, we sought to determine the influence of standard laboratory handling of microbes on within-strain genetic diversity and explore how these changes influence virulence characteristics and experimental outcomes. A single culture of S. pneumoniae D39 grown overnight resulted in the enrichment of previously rare genotypes present in bacterial freezer stocks and the introduction of new variation to the bacterial population through the acquisition of mutations. A comparison of D39 stocks from different laboratories demonstrated how changes in bacterial population structure taking place during individual culture events can cumulatively lead to fixed, divergent change that profoundly alters virulence characteristics. The passage of D39 through mouse models of infection, a process used to standardize virulence, resulted in the enrichment of high-fitness genotypes that were originally rare (<2% frequency) in D39 culture collection stocks and the loss of previously dominant genotypes. In the most striking example, the selection of a <2%-frequency genotype carrying a mutation in sdhB, a gene thought to be essential for the establishment of lung infection, was associated with enhanced systemic virulence. Three separately passaged D39 cultures originating from the same frozen stocks showed considerable genetic divergence despite comparable virulence. IMPORTANCE Laboratory bacteriology involves the use of high-density cultures that we often assume to be clonal but that in reality are populations consisting of multiple genotypes at various abundances. We have demonstrated that the genetic structure of a single population of a widely used Streptococcus pneumoniae strain can be substantially altered by even short-term laboratory handling and culture and that, over time, this can lead to changes in virulence characteristics. Our findings suggest that caution should be applied when comparing data generated in different laboratories using the same strain but also when comparing data within laboratories over time. Given the dramatic reductions in the cost of next-generation sequencing technology in recent years, we advocate for the frequent sampling and sequencing of bacterial isolate collections.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

Metal ions are required by all organisms for the chemical processes that support life. However, in excess they can also exert toxicity within biological systems. During infection, bacterial pathogens such as Streptococcus pneumoniae are exposed to host-imposed metal intoxication, where the toxic properties of metals, such as copper, are exploited to aid in microbial clearance. However, previous studies investigating the antimicrobial efficacy of copper in vivo have reported variable findings. Here, we use a highly copper-sensitive strain of S. pneumoniae, lacking both copper efflux and intracellular copper buffering by glutathione, to investigate how copper stress is managed and where it is encountered during infection. We show that this strain exhibits highly dysregulated copper homeostasis, leading to the attenuation of growth and hyperaccumulation of copper in vitro. In a murine infection model, whole-tissue copper quantitation and elemental bioimaging of the murine lung revealed that infection with S. pneumoniae resulted in increased copper abundance in specific tissues, with the formation of spatially discrete copper hot spots throughout the lung. While the increased copper was able to reduce the viability of the highly copper-sensitive strain in a pneumonia model, copper levels in professional phagocytes and in a bacteremic model were insufficient to prosecute bacterial clearance. Collectively, this study reveals that host copper is redistributed to sites of infection and can impact bacterial viability in a hypersusceptible strain. However, in wild-type S. pneumoniae, the concerted actions of the copper homeostatic mechanisms are sufficient to facilitate continued viability and virulence of the pathogen. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is one of the world's foremost bacterial pathogens. Treatment of both localized and systemic pneumococcal infection is becoming complicated by increasing rates of multidrug resistance globally. Copper is a potent antimicrobial agent used by the mammalian immune system in the defense against bacterial pathogens. However, unlike other bacterial species, this copper stress is unable to prosecute pneumococcal clearance. This study determines how the mammalian host inflicts copper stress on S. pneumoniae and the bacterial copper tolerance mechanisms that contribute to maintenance of viability and virulence in vitro and in vivo. This work has provided insight into the chemical biology of the host-pneumococcal interaction and identified a potential avenue for novel antimicrobial development.

Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin β1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.

Streptococcus pneumoniae is a common opportunistic pathogen that causes invasive pneumococcal disease (IPD), especially in children. This study aimed to determine the prevalence and molecular characteristics of S. pneumoniae isolated from children with IPD. A total of 78 S. pneumoniae isolates from aseptic body fluids of 70 IPD patients were collected at the Children's Hospital of Nanjing Medical University (Jiangsu Province, China) during 2017-2021. Whole-genome sequencing technology was used to analyze the serotype, sequence type (ST), virulence, and antibiotic resistance of the 78 invasive S. pneumoniae clinical isolates. Our results showed that the pneumococcal infection rate declined after the COVID-19 outbreak in 2019. Serotypes 19F, 14, 6A, 23F, 19A, and 6B were the most common strains. The pneumococcal conjugate vaccine (PCV) 13 serotype coverage rate was 87.1%. All isolates were classified by multi-locus sequence typing (MLST) analysis into 27 different STs, including 3 novel STs (ST17941, ST17942, and ST17944) and 1 novel allele [recP (558)]. The most predominant ST was ST271, followed by ST320 and ST876. All isolates carried the following virulence genes: cbpG, lytB, lytC, pce (cbpE), pavA, slrA, plr (gapA), hysA, nanA, eno, piuA, psaA, cppA, iga, htrA (degP), tig (ropA), zmpB, and ply. All isolates were multidrug resistant and had high levels of resistance to macrolides, tetracyclines, and sulfonamides. Taken together, this study revealed extensive genetic diversity among S. pneumoniae isolates from a single Chinese hospital. Wearing masks, universal infant vaccination with PCV13, and the launch of recombinant protein vaccine development programs could reduce the burden of IPD in children. IMPORTANCE Invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae in children remains a global burden and should be given more attention due to the fact that the pneumococcal vaccine is not fully covered globally. The molecular epidemiological characteristics of S. pneumoniae are not so clear, especially in these years of COVID-19. In this study, we collected S. pneumoniae isolates from the aseptic body fluid of children with IPD from 2017 to 2021 in a tertiary children's hospital in China and revealed the extensive genetic diversity of these isolates. Most importantly, we first found that the rate of pneumococcal infection has declined since the COVID-19 outbreak in 2019, which means that wearing masks could reduce the transmission of S. pneumoniae. In addition, it was shown that universal infant vaccination with PCV13 seems essential for reducing the burden of IPD in children.

Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Due to the resistance of Streptococcus pneumoniae to β-lactams, macrolides, and tetracyclines, treatment alternatives have become increasingly limited worldwide. We aim to describe the characterization of erythromycin-resistant S. pneumoniae (ERSP) strains in northeastern China over a period of 20 years. A total of 1,240 ERSP strains were collected and classified into five groups based on the ages of the patients. Etest strips and Kirby-Bauer disk diffusion were performed for drug susceptibility testing. The capsule swelling test was used for capsule typing. The phenotype of drug resistance was detected by the erythromycin and clindamycin double-disk method. The ermB, ermTR, mefA, and tetM genes were detected by PCR. Among the 1,240 ERSP strains, 510 were invasive isolates, and 730 were noninvasive isolates. The results of drug susceptibility testing showed that the rates of resistance to penicillin, amoxicillin, cefotaxime, ceftriaxone, meropenem, tetracycline, trimethoprim-sulfamethoxazole, and chloramphenicol varied among the different age groups. 19F, 19A, 23F, 14, and 6B were the serotypes that were commonly found among ERSP strains. Among all strains, 99.03% (1,228/1,240) exhibited an MLSB (macrolide-lincosamide-streptogramin B) resistance phenotype, of which 1,221 strains displayed a constitutive MLSB (cMLSB) phenotype and 7 strains showed an inducible MLSB (iMLSB) phenotype. All of these strains carried the ermB gene. In contrast, only 0.97% of strains of M phenotypes were found to carry the mefA gene. Both the ermB and mefA genes were detected in 704 strains that exhibited multidrug resistance, whereas the ermTR gene was not detected. Furthermore, 1,185 tetracycline-resistant strains were found to carry the tetM gene. Macrolide antimicrobial drugs should be used cautiously for the empirical treatment of S. pneumoniae infections. IMPORTANCE This study presents a retrospective analysis using 1,240 clinical erythromycin-resistant Streptococcus pneumoniae (ERSP) isolates collected in northeastern China between January 2000 and December 2019. The serotype distribution, corresponding vaccine coverage, as well as resistance phenotypes, genes, and mechanisms to macrolide and tetracycline of these isolates were systematically described, analyzed, and discussed. We hope that this study will inform clinicians in their respective regions when selecting antimicrobial agents. We also hope that this study is useful for researchers in related fields. Finally, we emphasize in this study that vaccination is the best preventive measure for S. pneumoniae infection considering its resistance to commonly used antibiotics. The determination of the S. pneumoniae serotype distribution also provides valuable empirical evidence for local health authorities when introducing appropriate vaccines in a specific area.

We characterized Streptococcus pneumoniae serotype 24F sequence type (ST) 162 isolated from Japanese children with invasive pneumococcal disease (IPD). Owing to its highly invasive nature, serotype 24F is expected to be isolated from clinically significant cases. Serotype 24F ST162 isolates tested in the present study did not grow at 35°C in ambient air. Therefore, antimicrobial susceptibility testing using the broth microdilution method, which is usually conducted in ambient air, cannot be performed, posing a clinical challenge. Clinical practitioners and laboratory personnel should be aware of the epidemiological, bacteriological, and molecular characteristics of serotype 24F ST162. We believe that our findings can help diagnose and treat IPD caused by serotype 24F ST162, a serotype expected to become problematic in the post-13 valent pneumococcal conjugate vaccine era.

Biofilm formation by Streptococcus pneumoniae is associated with colonization of the upper respiratory tract, including the carrier state, and with chronic respiratory infections in patients suffering from chronic obstructive pulmonary disease (COPD). The use of antibiotics alone to treat recalcitrant infections caused by biofilms is insufficient in many cases, requiring novel strategies based on a combination of antibiotics with other agents, including antibodies, enzybiotics, and antioxidants. In this work, we demonstrate that the third-generation oral cephalosporin cefditoren (CDN) and the antioxidant N-acetyl-l-cysteine (NAC) are synergistic against pneumococcal biofilms. Additionally, the combination of CDN and NAC resulted in the inhibition of bacterial growth (planktonic and biofilm cells) and destruction of the biofilm biomass. This marked antimicrobial effect was also observed in terms of viability in both inhibition (prevention) and disaggregation (treatment) assays. Moreover, the use of CDN and NAC reduced bacterial adhesion to human lung epithelial cells, confirming that this strategy of combining these two compounds is effective against resistant pneumococcal strains colonizing the lung epithelium. Finally, administration of CDN and NAC in mice suffering acute pneumococcal pneumonia caused by a multidrug-resistant strain was effective in clearing the bacteria from the respiratory tract in comparison to treatment with either compound alone. Overall, these results demonstrate that the combination of oral cephalosporins and antioxidants, such as CDN and NAC, respectively, is a promising strategy against respiratory biofilms caused by S. pneumoniae. IMPORTANCE Streptococcus pneumoniae is one of the deadliest bacterial pathogens, accounting for up to 2 million deaths annually prior to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccines have decreased the burden of diseases produced by S. pneumoniae, but the rise of antibiotic-resistant strains and nonvaccine serotypes is worrisome. Pneumococcal biofilms are associated with chronic respiratory infections, and treatment is challenging, making the search for new antibiofilm therapies a priority as biofilms become resistant to traditional antibiotics. In this work, we used the combination of an antibiotic (CDN) and an antioxidant (NAC) to treat the pneumococcal biofilms of relevant clinical isolates. We demonstrated a synergy between CDN and NAC that inhibited and treated pneumococcal biofilms, impaired pneumococcal adherence to the lung epithelium, and treated pneumonia in a mouse pneumonia model. We propose the widely used cephalosporin CDN and the repurposed drug NAC as a new antibiofilm therapy against S. pneumoniae biofilms, including those formed by antibiotic-resistant clinical isolates.

Multidrug resistance in Streptococcus pneumoniae (or pneumococcus) continues to be a global challenge. An important class of antibiotic resistance determinants disseminating in S. pneumoniae are >20-kb Tn916-related integrative and conjugative elements (ICEs), such as Tn2009, Tn6002, and Tn2010. Although conjugation has been implicated as the transfer mechanism for ICEs in several bacteria, including S. pneumoniae, the molecular basis for widespread dissemination of pneumococcal Tn916-related ICEs remains to be fully elucidated. We found that Tn2009 acquisition was not detectable via in vitro transformation nor conjugative mating with donor GA16833, yielding a transfer frequency of <10-7. GA16833 Tn2009 conjugative gene expression was not significantly induced, and ICE circular intermediate formation was not detected in biofilms. Consistently, Tn2009 transfer efficiency in biofilms was not affected by deletion of the ICE conjugative gene ftsK. However, GA16833 Tn2009 transfer occurred efficiently at a recombination frequency (rF) of 10-4 in dual-strain biofilms formed in a human nasopharyngeal cell bioreactor. DNase I addition and deletions of the early competence gene comE or transformation apparatus genes comEA and comEC in the D39 recipient strain prevented Tn2009 acquisition (rF of <10-7). Genome sequencing and single nucleotide polymorphism analyses of independent recombinants of recipient genotype identified ~33- to ~55-kb donor DNAs containing intact Tn2009, supporting homologous recombination. Additional pneumococcal donor and recipient combinations were demonstrated to efficiently transfer Tn916-related ICEs at a rF of 10-4 in the biofilms. Tn916-related ICEs horizontally disseminate at high frequency in human nasopharyngeal S. pneumoniae biofilms by transformation and homologous recombination of >30-kb DNA fragments into the pneumococcal genome. IMPORTANCE The World Health Organization has designated Streptococcus pneumoniae as a priority pathogen for research and development of new drug treatments due to extensive multidrug resistance. Multiple strains of S. pneumoniae colonize and form mixed biofilms in the human nasopharynx, which could enable exchange of antibiotic resistance determinants. Tn916-related integrative and conjugative elements (ICEs) are largely responsible for the widespread presence of macrolide and tetracycline resistance in S. pneumoniae. Utilizing a system that simulates colonization of donor and recipient S. pneumoniae strains in the human nasopharynx, efficient transfer of Tn916-related ICEs occurred in human nasopharyngeal biofilms, in contrast to in vitro conditions of planktonic cells with exogenous DNA. This high-frequency Tn916-related ICE transfer between S. pneumoniae strains in biofilms was due to transformation and homologous recombination, not conjugation. Understanding the molecular mechanism for dissemination of Tn916-related ICEs can facilitate the design of new strategies to combat antibiotic resistance.

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) is a human nasopharyngeal commensal, and host N-glycan metabolism promotes its colonization and invasion. It has been reported that glucose represses, while fetuin, a glycoconjugated model protein, induces, the genes involved in N-glycan degradation through the two-component system TCS07. However, the mechanisms of glucose repression and TCS07 induction remain unknown. Previously, we found that the pneumococcal aquaglyceroporin Pn-AqpC facilitates oxygen uptake, thereby contributing to the antioxidant potential and virulence. In this study, through Tandem Mass Tag (TMT) quantitative proteomics, we found that the deletion of Pn-aqpC caused a marked upregulation of 11 proteins involved in N-glycan degradation in glucose-grown pneumococcus R6. Both quantitative RT-PCR and GFP fluorescence reporters revealed that the upregulation of N-glycan genes was completely dependent on response regulator (RR) 07, but not on the histidine kinase HK07 of TCS07 or the phosphoryl-receiving aspartate residue of RR07 in ΔPn-aqpC, indicating that RR07 was activated in an HK07-independent manner when Pn-AqpC was absent. The deletion of Pn-aqpC also enhanced the expression of pyruvate formate lyase and increased formate production, probably due to reduced cellular oxygen content, indicating that a shunt of glucose catabolism to mixed acid fermentation occurs. Notably, formate induced the N-glycan degradation genes in glucose-grown R6, but the deletion of rr07 abolished this induction, indicating that formate activates RR07. However, the induction of N-glycan degradation proteins reduced the intraspecies competition of R6 in glucose. Therefore, although N-glycan degradation promotes pneumococcal pathogenesis, the glucose metabolites-based RR07 regulation reported here is of importance for balancing growth fitness and the pathogenicity of pneumococcus. IMPORTANCE Pneumococcus, a human opportunistic pathogen, is capable of metabolizing host complex N-glycans. N-glycan degradation promotes pneumococcus colonization in the nasopharynx as well as invasion into deeper tissues, thus significantly contributing to pathogenesis. It is known that the two-component system 07 induces the N-glycan metabolizing genes; however, how TCS07 is activated remains unknown. This study reveals that formate, the anaerobic fermentation metabolite of pneumococcus, is a novel activator of the response regulator (RR) 07. Although the high expression of N-glycan degradation genes promotes pneumococcal colonization in the nasopharynx and pathogenesis, this reduces pneumococcal growth fitness in glucose as indicated in this work. Notably, the presence of Pn-AqpC, an oxygen-transporting aquaglyceroporin, enables pneumococcus to maintain glucose homolactic acid fermentation, thus reducing formate production, maintaining RR07 inactivation, and controlling N-glycan degrading genes at a non-induced status. Thus, this study highlights a novel fermentation metabolism pattern linking TCS-regulated carbohydrate utilization strategies as a trade-off between the fitness and the pathogenicity of pneumococcus.

Streptococcus pneumoniae (Spn) is the world's leading cause of lower respiratory tract infection morbidity and mortality in children. However, current clinical microbiological methods have disadvantages. Spn can be difficult to grow in laboratory conditions if a patient is pre-treated, and Spn antigen testing has unclear clinical utility in children. Syndromic panel testing is less cost-effective than targeted PCR if clinical suspicion is high for a single pathogen. Also, such testing entails a full, expensive validation for each panel target if used for multiple respiratory sources. Therefore, better diagnostic modalities are needed. Our study validates a multiplex PCR assay with three genomic targets for semi-quantitative and quantitative Spn molecular detection from lower respiratory sources for clinical testing and from upper respiratory sources for research investigation.

Streptococcus pneumoniae (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).

Streptococcus pneumoniae (Spn) colonizes the lungs, killing millions every year. During its metabolism, Spn produces abundant amounts of hydrogen peroxide. When produced in the lung parenchyma, Spn-hydrogen peroxide (H2O2) causes the death of lung cells, and details of the mechanism are studied here. We found that Spn-H2O2 targets intracellular proteins, resulting in the contraction of the cell cytoskeleton and disruption of mitochondrial function, ultimately contributing to cell death. Intracellular proteins targeted by Spn-H2O2 included cytochrome c and, surprisingly, a protein of the cell cytoskeleton, beta-tubulin. To study the details of oxidative reactions, we used, as a surrogate model, the oxidation of another hemoprotein, hemoglobin. Using the surrogate model, we specifically identified a highly reactive radical whose creation was catalyzed by Spn-H2O2. In sum, we demonstrated that the oxidation of intracellular targets by Spn-H2O2 plays an important role in the cytotoxicity caused by Spn, thus providing new targets for interventions.

Pet bite-related infections are commonly caused by the pet's oral flora transmitted to the animal handlers through the bite wounds. In this study, we isolated a streptococcus, HKU75T, in pure culture from the purulent discharge collected from a guinea pig bite wound in a previously healthy young patient. HKU75T was alpha-hemolytic on sheep blood agar and agglutinated with Lancefield group D and group G antisera. API 20 STREP showed that the most likely identity for HKU75T was S. suis I with 85.4% confidence while Vitek 2 showed that HKU75T was unidentifiable. MALDI-TOF MS identified HKU75T as Streptococcus suis (score of 1.86 only). 16S rRNA gene sequencing showed that HKU75T was most closely related to S. parasuis (98.3% nucleotide identity), whereas partial groEL and rpoB gene sequencing showed that it was most closely related to S. suis (81.8% and 89.8% nucleotide identity respectively). Whole genome sequencing and intergenomic distance determined by ANI revealed that there was <85% identity between the genome of HKU75T and those of all other known Streptococcus species. Genome classification using concatenated sequences of 92 bacterial core genes showed that HKU75T belonged to the Suis group. groEL gene sequences identical to that of HKU75T could be directly amplified from the oral cavities of the two guinea pigs owned by the patient. HKU75T is a novel Streptococcus species, which we propose to be named S. oriscaviae. The oral cavity of guinea pigs is presumably a reservoir of S. oriscaviae. Some of the reported S. suis strains isolated from clinical specimens may be S. oriscaviae. IMPORTANCE We reported the discovery of a novel Streptococcus species, propose to be named Streptococcus oriscaviae, from the pus collected from a guinea pig bite wound in a healthy young patient. The bacterium was initially misidentified as S. suis/S. parasuis by biochemical tests, mass spectrometry. and housekeeping genes sequencing. Its novelty was confirmed by whole genome sequencing. Comparative genomic studies showed that S. oriscaviae belongs to the Suis group. S. oriscaviae sequences were detected in the oral cavities of the two guinea pigs owned by the patient, suggesting that the oral cavity of guinea pigs could be a reservoir of S. oriscaviae. Some of the reported S. suis strains may be S. oriscaviae. Further studies are warranted to refine our knowledge on this novel Streptococcus species.